The polymerase chain reaction (PCR) is a method to selectively amplify DNA. The method uses paired sets of oligonucleotides that hybridise to opposite strands of DNA and define the limits of the sequence that is amplified. The oligonucleotides prime multiple sequential rounds of DNA synthesis catalysed by a thermostable DNA polymerase. Each round of synthesis is normally preceded by a melting and re-annealing step. The method can rapidly amplify virtually any DNA sequence (Saiki et al., Science 239:487, 1988).
PCR is widely used for the genetic identification of unique sequences in individual organisms. Uses include: forensic analysis (Gill, (2002) Biotechniques 32, 366-372), diagnosis of genetic disorders and disease susceptibility, neoplastic disease (Raj, (1998) Cancer 82, 1419-1442), detection of infectious diseases (Daxboeck (2003) Clinical Microbiology and Infection 9, 263-273) and food testing (Malorne (2003) International Journal of Food Microbiology 83, 39-48). The DNA of interest is typically amplified from genomic DNA, viral DNA, or from cDNA reverse transcribed from RNA.
In common with other assays, PCR is subject to both ‘false negative’ and ‘false positive’ results. False negative results are due to reaction failure. False positive results may be caused by primers annealing to sequences other than the true recognition sequence leading to amplification of spurious products, or by primers annealing to the true recognition sequence present in contaminating DNA derived from a source other than the sample being diagnosed. True recognition sequences contained in the sample being diagnosed will be termed “target” in the following. True recognition sequences contained in contaminating DNA are not classified as target DNA.
The most frequent and potent source of contaminating DNA that causes false positives is previously amplified PCR products (termed amplicons) with recognition sequences identical to those of the primers being used (Rolfs et al., (1992) PCR: clinical diagnostics and research. Springer-Verlag, Berlin). The probability of contamination increases when diagnostic PCR is carried out many times for one DNA sequence, and when the PCR technique has been designed to detect a small number of molecules of DNA. Contamination by previous PCR products is called ‘carryover’ to distinguish it from contamination by DNA from other sources.
Even strict adherence to good laboratory practice and protocols that aim to avoid any contact of amplicon with pre-amplification reagents or samples cannot guarantee the absence of false positives due to amplicon contamination. As a result, a significant part of the cost of diagnostic or forensic PCR assays is caused by the need to include a relatively large number of negative controls and to repeat arrays of assays if the slightest indication of a contamination is found.
Thus, a method to individually ascertain the absence of amplicon contamination in any reaction is highly desirable.
Two approaches can be envisioned to solve the general problem posed by amplicon contamination: either the amplicon can be destroyed subsequent to its detection in order to avoid it contaminating any later reaction, or methods must be found to discriminate amplification product arising from DNA as opposed to amplicon contamination. The former idea has been implemented in the method of dUTP incorporation and subsequent destruction of the amplicon by uracil-N-glycosylase (see EP0401037 and references contained therein; Longo et al. (1990), Gene 93, 125-128), a method widely employed today. Other methods have been devised that follow similar rationales (Cimino et al. (1991), Nucleic Acids Research 19, 773-774; Walder et al. (1993), Nucleic Acids Research 21, 4339-434).
Richards (U.S. Pat. No. 5,650,302) discloses a method to incorporate restriction nuclease recognition sites into the primers in order to render amplicon contaminants un-amplifiable when digested with the correspondent nuclease prior to amplification.
Destruction of the amplicon after determination of its presence or quantity will reduce the likelihood of downstream contamination, but since the amplicon destruction itself is a process subject to possible failure, it cannot positively rule out false positive results due to amplicon contamination.
Shuber (U.S. Pat. No. 6,207,372) discloses a multiple duplex primer PCR method, where a universal primer sequence at the 5′ end of various primer pairs allows for uniform amplification conditions for multiple targets.
Shuldiner (WO9115601) discloses a method by which RT-PCR-reactions, where the first step is elongation of a DNA primer on a RNA template, can be made more specific over a background of possible DNA contaminant sequences. This method employs two primers, one of which is a hybrid sequence comprising a target-RNA-specific sequence tract and a tagging-tract. Discrimination is achieved between target RNA and possible contaminating genomic or plasmid DNA on the basis of the different hybridization temperatures of DNA-DNA versus RNA-DNA duplexes. Since RNA-DNA-double strands are more stable and hence, have a higher melting or annealing temperature than DNA-DNA double strands, a reaction temperature can be selected at which the target-specific primer part will only anneal to a RNA target. Subsequent duplication of this first DNA transcript generated from the hybrid primer will result in a DNA strand that is elongated at its 3′ end with the complementary tagging sequence, to which the hybrid primer will anneal in all subsequent amplification steps at the elevated temperature. This means that the method disclosed in WO9115601 cannot discriminate between, or exclude from being amplified, DNA amplicon produced in a previous reaction using the same primer set, although it may be a useful tool to exclude amplification from genomic DNA contaminations.
In parallel to any efforts to reduce the occurrence of contamination, it is desired to be able to discern amplification product arising from target sequences in the sample (the true positive result) and the false positive result arising from amplicon contamination.
Shuber (WO 9920798) has disclosed a method to detect contamination by amplicon sequences that relies on the use of two different oligonucleotide primer sets in two different amplification reactions. A first set of primers comprising a target-detection sequence and a contamination-detection-sequence, which is added to the 5′ end of the primers, are employed to detect the presence of target sequence in the original sample, a reaction that is termed “first amplification reaction”. In a second reaction mixture, a second set of primers comprising only the contamination detection sequence, are used on the original sample, in order to detect contamination of the sample by amplicon molecules produced in previous reactions.
The method disclosed in WO 9920798 achieves detection of amplicon contamination in the original sample, however it does not rule out positively the presence of amplicon contamination in any step that may be specific for the “first” (according to the terminology of WO 9920798) amplification reaction. As one example, the two reactions differ in the primers employed, and thus any amplicon contamination in the solution containing the oligonucleotide primers for the first reaction would not be detected.
Another aspect of the disclosure of WO 9920798 that may be improved upon is the use of two reaction vessels. Although tolerable in some instances, the doubling of the expense in time and material both for the reaction preparation and reagents may offset the advantage in savings from reducing false positives. A method that enables the discrimination of true and false positives without the expense of having to conduct two separate reactions would thus be highly desirable.